Vav1 plays an essential role in T cell development and TCR-induced activation, and is required for TCR capping, actin cytoskeleton reorganization, and immunological synapse (IS) formation in T cells. T cells express two additional Vav family members, Vav2 and Vav3, but their precise role in T cell development or activation is unknown. Our past work focused on several aspects of the regulation and function of Vav1 in T cells. We will extend these studies and address several unresolved aspects of the function of Vav in TCR signaling pathways, with increased emphasis on the use of antigen-specific primary T cells and reconstitution studies in Vav-deficient T cells. We will use a combination of biochemical and genetic approaches to analyze Vav-dependent protein-protein interactions and signaling pathways leading to actin cytoskeleton reorganization, CD69 up-regulation, and activation of MAP kinases or transcription factors. In Aim 1, we will map the physiologically relevant tyrosine phosphorylation sites in Vav1, identify the responsible PTKs, and determine how phosphorylation at different tyrosine residues differentially regulates distinct Vav-induced signals. In Aim 2, we will explore functional interaction between Vav and the Ras pathway, and attempt to identify signaling components that mediate cross talk between these two pathways. In Aim 3, we will explore the mechanism that links Vav1 to AP-1 activation by analyzing the role of Vav1 in transcriptional and post-transcriptional events that regulate AP-1 components, with special emphasis on MEF2 transcription factors as potentially novel mediators of Vav1 function. In Aim 4, we will examine the working hypothesis that actin cytoskeleton reorganization initiated by Vav1 and Rho family GTPase is an important driving force in assembly of the IS and lipid raft clustering. Lastly, in Aim 5, we will analyze the respective roles of Vav1, -2 and -3 in T cell signaling pathways. These studies will clarify the role of Vav1 in different TCRJCD28 signaling pathways, improve our understanding of its regulation and coupling to distinct downstream signaling events, and elucidate the role of Vav2 and Vav3 in T cell activation.